Real-Time PCR Instrument Calibration and Verification Kits

Spend your time on research, not on instrument maintenance

Quickly and easily calibrate and verify the performance of your Applied Biosystems real-time PCR system using our instrument-specific kits and plates.

 

Applied Biosystems spectral calibration kits establish the pure dye spectra and multicomponent values (standards) for each reference dye. These standards enable the real-time PCR system to collect pure spectral information, which it uses algorithmically to help ensure accurate data analysis. Depending on which Applied Biosystems instrument you have, there are plate options for individual dye calibration or multi-dye plates that enable simultaneous calibration, which can greatly reduce the time and effort spent on maintenance.

 

Confirm the performance of your Applied Biosystems instrument with a TaqMan RNase P instrument verification plate. These plates are pre-loaded with real-time PCR reagents and the TaqMan Assay for the single-copy RNase P gene. Each well contains PCR master mix, RNase P primers, FAM dye-labeled probe, and a known concentration of human genomic DNA template.

 

Always consult your instrument user guide for the recommended calibration/verification schedule and instructions.

Calibration kits and verification plates by instrument model

Types of calibration

Our calibration kits and plates support several types of calibration.

Region of interest (ROI) calibration

ROI calibration maps the positions of the wells on the sample block of the instrument. The software uses the ROI calibration data to associate increases in fluorescence during a run with the wells on the plate or array card. The instrument uses a set of optical filters to distinguish the fluorescence emissions gathered during runs. The software generates a calibration image for each filter to account for minor differences in the optical path.

Successful ROI calibration

During the ROI calibration, the instrument software captures images of the ROI calibration plate or array card at each instrument filter. An ROI calibration passes if the collected image for each filter distinguishes all wells of the ROI plate or array card. Each well in the image must be distinct and visible at the same luminosity relative to the other wells in the image.

Background calibration

Background calibration captures a background image for each optical filter in the absence of sample and reagent and compares the fluorescence from each well to the average for the plate. The software uses that data during subsequent experiments to remove background fluorescence from the experimental data.

Successful background calibration

During the background calibration, the software captures a series of images of the background plate or array card using each instrument filter. The software compares the fluorescence from each well to the average for the plate or array card. A background calibration passes if the collected images for all filters are free of abnormal fluorescence.

Uniformity calibration

Uniformity calibration generates data that allows the software to compensate for the physical effects (non-uniformity of the optical system such as light source) of the instrument filters.

Successful Uniformity calibration

During the uniformity calibration, the software captures a series of images of the ROI plate or array card using each instrument filter. After the calibration, you can review the data. The Analysis Data plot displays the fluorescence data in all filters. A uniformity calibration passes if signals from each well follows a uniform trend.

Dye calibration

Dye calibration collects calibration data generated from the dye standards to characterize and distinguish the individual contribution of each dye in the total fluorescence collected by the instrument. After each run, the instrument software receives data in the form of a raw spectra signal for each reading. The software determines the contribution of the fluorescent dyes used in each reaction by comparing the raw spectra to the pure spectra calibration data. When you save an experiment after analysis, the instrument software stores the pure spectra with the collected fluorescence data for that experiment.

Successful Dye calibration

During the dye calibration, the software extracts the dye calibration data and compares the fluorescence signal generated by each well to the collective spectra for the entire calibration plate or array card. Dye spectra are generally acceptable if they peak within the same filter as their group but diverge slightly at other wavelengths.

Calibration helps enable effective multiplexing

To enable success when multiplexing (which helps save on both samples and costs), our custom dye calibration plates help ensure you realize the same level of sensitivity when running multiple targets in a single well as you would running a just one (singleplex). This gene quantification experiment demonstrates comparable results between reactions performed in individual tubes and in 4-plex reactions.

Comparison of singleplex and multiplex gene expression assays

Series of data images comparing singleplex and multiplex gene expression assays
Series of data images comparing singleplex and multiplex gene expression assays
Series of data images comparing singleplex and multiplex gene expression assays
Series of data images comparing singleplex and multiplex gene expression assays

Comparison of singleplex and multiplex gene expression assays. Comparison of singleplex and multiplex gene expression assays. (A) EGFR assay, FAM dye; (B) BRCA1 assay, VIC dye; (C) ESR1 assay, JUN dye; (D) RNase P assay, ABY dye. Amplification was performed on the QuantStudio 7 Real-Time PCR System using TaqMan Multiplex Master Mix. The figure shows amplification plots (left) and linear curves (right) for 4 assays amplified in singleplex (blue) and 4-plex reactions (red) in a dilution series from 20,000 pg to 2 pg of reference colon cDNA per 10 μL reaction. Average Ct value (lines) and average standard deviation (bars) for the dilution series are represented in their respective graphs and show the concordance between singleplex and 4-plex reactions. PCR efficiencies for each target in both single- and multiplex reactions are shown here:

 

 

EGFR

BRCA1

ESR1

RNase P

Singleplex

96.09%

93.56%

97.13%

96.91%

Multiplex (4 targets)

96.39%

94.93%

95.81%

98.1%

For Research Use Only. Not for use in diagnostic procedures.